Determination of serum cortisol fractions by isocolloidosmolar equilibrium dialysis. Changes during pregnancy.

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Abstract
A modified equilibrium dialysis method to determine the percentage of protein-unbound cortisol in the total serum cortisol (unbound cortisol percent) was devised. When serum was dialyzed against an ordinary buffer solution, unbound cortisol percent increased with the time of dialysis, accompanying a progressive decrease in serum protein concentration. These changes seemed to be due to the difference of colloidal osmotic pressure between the serum and the buffer. An addition of 3% dextran in the buffer resulted in a stable equilibrium and both the unbound cortisol percent and the protein concentration stayed unchanged after 12 h of the dialysis. The determination of unbound cortisol percent was carried out by dialyzing serum against 3% dextran buffer for 18 h and the method was called an isocolloidosmolar equilibrium dialysis. Dextran did not bind cortisol at all. Additions of graded amounts of cortisol in the dialysate caused significant increases in unbound cortisol percent of a pooled serum. The ratio of unbound cortisol to albumin-bound cortisol was determined by the same dialysis system using heat-treated serum in the presence of nonradioactive cortisol. A combination of these 2 determinations enables assessment of concentrations of unbound, albumin-bound and transcortin-bound cortisol in serum. Both intra-assay and inter-assay precisions of these methods were excellent (at most .+-. 4.65%). In normal subjects (cortisol concentration: 7.1 .+-. 2.5 .mu.g/100 ml), 5.8 .+-. 0.7% was present as unbound cortisol, 15.8 .+-. 2.5% as albumin-bound and 78.3 .+-. 2.8% as transcortin-bound cortisol. Concentrations of all serum cortisol fractions increased with the course of pregnancy, however, relative increment of transcortin-bound cortisol was larger than that of total cortisol, while those of unbound and albumin-bound cortisol were far less. Percentages of transcortin-bound and albumin-bound cortisol changed in parallel to changes in concentrations of these cortisol-binding proteins during pregnancy. The method requires a small amount of serum and is applicable to determine three cortisol fractions in individual sera.